JP2021156901A - Method for determining molecular probe - Google Patents

Abstract

To provide a simpler and more accurate method for determining a molecular probe for a target compound.SOLUTION: A method for determining a molecular probe according to the embodiment is a method for determining a molecular probe for capturing a target compound. The method includes: a step S1 in which the target compound is brought into contact with one type of candidate molecules and an obtained mixture is electrophoresed on gel; and a step S2 in which, when the candidate molecule is separated into a plurality of bands on the gel after electrophoresis, the candidate molecule is determined as the molecular probe for capturing the target compound.SELECTED DRAWING: Figure 1

Description

Embodiments of the present invention relate to a method for determining a molecular probe.

In the field of compound detection, molecular probes that selectively or specifically capture the target compound to be detected are often used. The molecular probe can, for example, label or separate the target compound, which is useful for efficient detection and separation of the target compound.

Therefore, the optimum molecular probe for the target compound is being searched for every day. Molecular probes can be found, for example, by investigating whether various candidate molecules bind to the target compound. As such a method, for example, electrophoresis or column chromatography is used.

In the method using electrophoresis, the target compound is brought into contact with various candidate molecules for electrophoresis. Then, the candidate molecule bound to the target compound is determined as a molecular probe for capturing the target compound by utilizing the fact that the migration distance changes with the candidate molecule not bound to the target compound.

In the method using column chromatography, for example, the target compound is fixed on the solid phase of the column, and the candidate molecule which is the liquid phase is allowed to flow on the column. Then, the candidate molecule that binds to the target compound and remains in the solid phase is determined as the molecular probe of the target compound.

On the other hand, detecting low molecular weight compounds is very useful in various fields such as drug investigation, doping test, and medical diagnosis. However, since a low molecular weight compound has a small molecular weight and a simple structure, it is very difficult to distinguish it from other compounds having a similar structure. Therefore, the development of a highly sensitive detection method for low molecular weight compounds is required.

An object of the present invention is to provide a simpler and more accurate method for determining a molecular probe of a target compound.

The method for determining a molecular probe according to an embodiment is a method for determining a molecular probe that captures a target compound. In the method, (S1) one kind of candidate molecule is brought into contact with a target compound, and the obtained mixture is electrophoresed on a gel, and (S2) a plurality of bands of candidate molecules are formed on the gel after electrophoresis. When separated into, it involves determining the candidate molecule as a molecular probe that captures the target compound.

FIG. 1 is a flowchart showing a method for determining a molecular probe according to an embodiment. FIG. 2 is a schematic view showing the steps of the molecular probe determination method of the embodiment. FIG. 3 is a flowchart showing a method for determining a molecular probe according to the embodiment. FIG. 4 is a schematic view showing the steps of the molecular probe determination method of the embodiment. FIG. 5 is a flowchart showing a method for determining a molecular probe according to the embodiment. FIG. 6 is a schematic view showing the steps of the molecular probe determination method of the embodiment. FIG. 7 is a flowchart showing a method for determining a molecular probe according to the embodiment. FIG. 8 is a flowchart showing a method for determining a molecular probe according to the embodiment. FIG. 9 is a schematic diagram illustrating how to determine the molecular probe of the embodiment. FIG. 10 is a schematic diagram illustrating how to determine the molecular probe of the embodiment.

Hereinafter, various embodiments will be described with reference to the drawings. Each figure is a schematic diagram for facilitating the understanding of the embodiment, and the shape, dimensions, ratio, etc. may differ from the actual ones. can do.

The method for determining a molecular probe according to the first embodiment is a method for determining a molecular probe that captures a target compound. In the method, (S1) one kind of candidate molecule is brought into contact with a target compound, and the obtained mixture is electrophoresed on a gel, and (S2) a plurality of bands of candidate molecules are formed on the gel after electrophoresis. When separated into, it involves determining the candidate molecule as a molecular probe that captures the target compound. According to the second and third embodiments, a method for determining a molecular probe from a plurality of candidate molecules is provided.

Hereinafter, a method for determining the molecular probe according to the first to third embodiments will be described.

(First Embodiment)
FIG. 1 is a diagram showing a schematic flow of a method for determining a molecular probe according to an embodiment. In the method, (S1) one kind of candidate molecule is brought into contact with a target compound, and the obtained mixture is electrophoresed on a gel, and (S2) a plurality of bands of candidate molecules are formed on the gel after electrophoresis. If separated into, or if the band is widened, it involves determining the candidate molecule as a molecular probe that captures the target compound.

Hereinafter, the principle that a molecular probe that captures a target compound can be determined by performing each of the above steps will be described with reference to FIG. FIG. 2 is a schematic diagram showing a process of a method for determining a molecular probe.

First, a solution 4 containing one type of candidate molecule 3 and a target compound 5 are added to a recess 2 formed at one end of the first gel 1 shown in FIG. 2 (a). As a result, the candidate molecule 3 and the target compound 5 are brought into contact with each other ((b) in FIG. 2). Candidate molecule 3 can also be pre-labeled for optical observation on the gel.

Next, the first gel 1 is electrophoresed. The migration direction of the electrophoresis is the direction from the end to which the solution 4 is added to the other end of the first gel 1.

As shown in FIG. 2C, when the candidate molecule 3 is a molecule that binds to the target compound 5, the candidate molecule 3 is separated into two bands 6a and 6b on the first gel 1. Alternatively, the band spreads in the flow direction. The reason why the band is separated into two or spreads in this way will be described below.

The candidate molecule 3 used is one type, and a plurality of the candidate molecules 3 of this one type are contained in the solution 4. When the candidate molecule 3 is a molecule having a binding ability to the target compound 5, not all the candidate molecules 3 bind to the target compound 5 when they come into contact with the target compound 5, but they bind to the target compound 5. There are those that form the complex 7 and those that do not bind to the target compound 5. Further, since the bond between the candidate molecule 3 and the target compound 5 is usually an equilibrium reaction that reversibly changes due to factors such as the structure, dissociation constant, or free energy change between the candidate molecule 3 and the target compound 5, the bond is formed. It is also possible to transition between states and uncoupled states. As described above, the existence of the candidate molecule 3 having a binding ability to the target compound 5 as an unbound state exists with a very high probability.

The complex 7 composed of the candidate molecule 3 and the target compound 5 and the candidate molecule 3 that does not bind to the target compound 5 have different isoelectric points, molecular weights, and / or higher-order structures. Therefore, they are separated as bands 6a and 6b, respectively. Alternatively, when candidate molecules 3 having different binding times with the target compound 5 are mixed, the separated bands are not clearly separated but spread in a broad form. Here, the band spread in this way is also referred to as a separated band.

Therefore, as shown in FIG. 2C, the candidate molecule 3 separated into a plurality of bands and the candidate molecule 3 in which the band is widened are regarded as molecules that bind to the target compound 5, and are molecular probes that capture the target compound 5. Can be determined.

On the other hand, when the candidate molecule 3 is a molecule that does not bind to the target compound 5 as shown in FIG. 2D, the candidate molecule 3 migrates as one band 6c and is not separated. In addition, the length of the band in the migration direction does not expand more than expected by free diffusion. In this case, it can be determined that the candidate molecule 3 is not a molecular probe that captures the target compound 5.

Furthermore, it is also possible to determine whether or not there is a difference in band separation or spread by comparing the result of electrophoresis of the candidate molecule 3 without contacting the target compound.

In the method described above, the candidate molecule 3 needs to be in a state where it can bind to the target compound 5 as a molecular probe during electrophoresis. Therefore, the electrophoresis is preferably performed by, for example, a method capable of maintaining the three-dimensional structure when the candidate molecule 3 is used as a molecular probe. Thereby, it is possible to inspect whether or not the candidate molecule 3 having the original three-dimensional structure that has not been denatured binds to the target compound 5, and it is possible to accurately determine the molecular probe that captures the target compound 5.

As such an electrophoresis method, isoelectric focusing (IEF) or native electrophoresis can be used. The native electrophoresis method may be, for example, a BN (Blueactive) electrophoresis method.

For example, if the target compound 5 is a charged compound, IEF can be used. When it is desired to determine a molecular probe whose three-dimensional structure is changed by binding of the target compound 5, it is preferable to use native electrophoresis. If the candidate molecule 3 is a peptide or protein and neither the target compound 5 nor the candidate molecule 3 has a charge or a small charge, BN electrophoresis can be used.

When using native electrophoresis or BN electrophoresis, continued electrophoresis causes the band to continue to move and migrate to the other end of the gel. Therefore, it is necessary to stop the electrophoresis at a desired time so that the band stops at an observable position. The time for electrophoresis varies depending on voltage, temperature, concentration, molecular weight, etc., but for example, electrophoresis can be performed over about 20 minutes to 2 hours.

The first gel 1 may be any gel that is usually used in the electrophoresis method used in the method of the embodiment. For example, the first gel 1 is a polyacrylamide gel, an agarose gel, or the like. The first gel 1 is selected according to the type of electrophoresis used. For example, when IEF is used, a gel having a pH gradient using the above gel as a material is used. Such a gel can be prepared at the same time as the gel is prepared by adding an amphoteric carrier (carrier amphoterite) to the gel and applying an electric field to form a pH gradient, or by using an acrylamide derivative having various IP side chains. It can be manufactured by a method of forming a gradient (IPG method) or the like.

Candidate molecule 3 is a molecule that is expected to be a molecular probe for target compound 5. Candidate molecule 3 is, for example, a protein, polypeptide, nucleic acid or aptamer.

Labeling of the candidate molecule 3 may be performed, for example, by a technique usually used in the electrophoresis method used in the method of the embodiment. For example, a fluorescent dye, a dye, a radioactive isotope, or the like may be used for the label. When the candidate molecule is a protein or peptide, for example, FITC can be used. FITC is a fluorescent molecule, and the candidate molecule can be labeled by reacting its isothiocyanate group with the N-terminal primary amine of a protein or peptide which is a candidate molecule. Alternatively, candidate molecule 3 which is a protein or peptide may be stained with silver after electrophoresis. When BN electrophoresis is used, it can be labeled by adsorbing Coomassie Brilliant Blue on a candidate molecule 3 which is a protein or peptide. With the markings as described above, the band can be observed, for example, visually, by a transilluminator, or by autoradiography.

Solution 4 is a solution in which the candidate molecule 3 is contained in an appropriate solvent. The solvent is, for example, distilled water, physiological saline or a buffer solution. Solution 4 contains one type of candidate molecule 3 and does not contain a substance that adversely affects the step of determining a molecular probe. Further, the solution 4 can have the same composition as the solvent when the candidate molecule 3 is used as a molecular probe.

The target compound 5 is, for example, a small molecule compound, a peptide, a protein, a glycoprotein, a virus, or an extracellular vesicle, and is an abusive drug, an explosive, a poison, a pesticide, an allergen, a mycotoxin, an odor component, a pathogen, or a cancer. It is a biomarker that suggests various diseases and health conditions such as. In particular, it is possible to obtain a high-performance molecular probe for low-molecular-weight compounds, which has been difficult to obtain in the past.

For example, the target compound 5 may be a substance contained in a liquid solvent. The liquid solvent is, for example, water, physiological saline, a buffer solution, or the like, and optionally contains an organic solvent or a surfactant. The liquid containing the target compound 5 has a composition that does not contain substances that adversely affect the process of determining the molecular probe. Further, the liquid containing the target compound 5 can have the same composition as the solvent when the candidate molecule 3 is used as a molecular probe.

When the target compound 5 is prepared as a liquid reagent, the solution 4 containing the candidate molecule 3 and the reagent are mixed in advance before the step (S2) and added to the recess 2 of the first gel 1. May be good. Alternatively, the reagent may be added to the recess 2 or the entire first gel 1 before or after the candidate molecule 3 is added to the recess 2 of the first gel 1.

Alternatively, the target compound 5 may be a substance contained in the gas solvent. The gaseous solvent is, for example, air, nitrogen, oxygen, hydrogen or carbon dioxide. The gas containing the target compound 5 has a composition that does not contain a substance that adversely affects the step of determining the molecular probe. Further, the gas containing the target compound 5 can have the same composition as the atmosphere of the environment in which the target compound 5 is being detected.

When the target compound 5 is prepared as a gaseous reagent, the reagent may be previously blown into the solution 4 containing the candidate molecule 3 to mix, and the obtained mixture may be added to the recess 2 of the first gel 1. Alternatively, after adding the candidate molecule 3 to the recess 2 of the first gel 1, the gel may be placed in a container filled with the gaseous reagent to perform electrophoresis while exposing the candidate molecule 3 to the target compound 5.

Further, after adding the candidate molecule 3 to the recess 2 of the first gel 1, the gel is placed in a container containing the solid or liquid target compound 5, and the candidate molecule 3 is volatile to be generated from the target compound 5. It is also possible to perform electrophoresis while exposing to a substance. In this case, even if some of the components of the target compound 5 are not volatile, the candidate molecule 3 that binds to the volatile substance (that is, the odor component) peculiar to the target compound 5 can be determined.

The method of the embodiment performs electrophoresis using one type of candidate molecule 3, and uses as an index that the candidate molecule is separated into a plurality of bands or the bands spread in the migration direction. Further, if necessary, it can be compared with the result of electrophoresis of the candidate molecule 3 which is not in contact with the target compound. Therefore, the candidate molecule 3 bound to the target compound can be easily identified.

Moreover, according to the determination method of the embodiment, it is not necessary to modify or fix the target compound. Therefore, the molecular probe can be selected with the original structure of the target compound 5, and the molecular probe of the target compound can be accurately determined. It is also possible to find a molecular probe that recognizes and binds to the entire target compound without modification or fixation. Moreover, the method of the embodiment is very simple to operate.

(Second Embodiment)
The method according to the second embodiment determines a molecular probe using a plurality of types of candidate molecules. FIG. 3 is a diagram showing a schematic flow of a method for determining a molecular probe according to the second embodiment. The method includes the following steps. (S11) Electrophoresis treatment of a plurality of types of candidate molecules on a gel (first electrophoresis), (S12) A mixture obtained by contacting a plurality of types of candidate molecules on the gel after the first electrophoresis with a target compound. Is electrophoresed in a direction orthogonal to the migration direction of the first migration (second migration), and (S13) is separated into a plurality of bands in the migration direction of the second migration on the gel after the second migration. To determine a candidate molecule or a candidate molecule whose band has expanded in the migration direction of the second electrophoresis as a molecular probe that captures the target compound.

Hereinafter, the principle that a molecular probe that captures a target compound can be determined by performing each of the above steps will be described with reference to FIG. FIG. 4 is a schematic view showing a process of the method of the second embodiment.

First, a solution 4 containing a plurality of types of candidate molecules 3 is prepared. Solution 4 contains five candidate molecules 3a, 3b, 3c, 3d and 3e.

First, the solution 4 is added to a recess (not shown) formed at one end of the first gel 1 shown in FIG. 4 (a) (FIG. 4 (b)). Then, it is electrophoresed (FIG. 4 (c)). This electrophoresis is called the first electrophoresis. The migration direction of the first electrophoresis is the direction from the end to which the solution 4 of the first gel 1 is added to the other end.

By the first electrophoresis, the candidate molecules 3a, 3b, 3c, 3d and 3e are separated by the difference in their isoelectric point, molecular weight, higher-order structure and the like according to the type of electrophoresis used. By this electrophoresis, the candidate molecules 3a, 3b, 3c, 3d and 3e are separated into bands 6a, 6b, 6c, 6d and 6e, respectively.

Next, as shown in FIG. 4 (e), the longitudinal side surface of the first gel 1 is bound to the end of the second gel 8, and then the target compound 5 is added to the first gel 1. The target compound 5 is brought into contact with the candidate molecules 3a, 3b, 3c, 3d and 3e ((d) in FIG. 4). Alternatively, the target compound 5 is added to the first gel 1 to bring the target compound 5 into contact with the candidate molecules 3a, 3b, 3c, 3d and 3e, and then the longitudinal side surface of the first gel 1 is seconded. It binds to the end of gel 8. Alternatively, the target compound 5 is added to the second gel 8 in advance, and the longitudinal side surface of the first gel 1 is bound to the end of the second gel 8. For example, a liquid reagent containing the target compound 5 is added dropwise to the first gel 1 or the second gel 8. Alternatively, a gas reagent containing the target compound 5 is filled in a container, and the first gel 1 and the second gel are placed in the container. Alternatively, the first gel 1 and the second gel are placed in a container containing the target compound 5. If necessary, for example, it may be left for several minutes to several tens of minutes so that the target compound 5 comes into sufficient contact with the candidate molecule 3. Alternatively, the leaving time may be determined according to the method when the candidate molecule 3 is used as a molecular probe. For example, if you want to detect a very small amount of target compound with high sensitivity over time, it is preferable to leave it for a sufficiently long time, and if you want to detect the target compound quickly, it is better to leave it for a short time. preferable.

Then, the electrophoresis process is performed in the direction orthogonal to the migration direction of the first electrophoresis. This electrophoresis is called the second electrophoresis. The migration direction of the second electrophoresis is the direction from the end to which the first gel 1 is bound to the other end of the second gel 8.

The second electrophoresis is performed using the same electrophoresis method as the first electrophoresis.

By the second migration, the candidate molecules 3a, 3b, 3c, 3d and 3e move on the second gel 8 in the migration direction, respectively. The bands 6a, 6c and 6e consisting of the candidate molecules 3a, 3c and 3e that do not bind to the target compound 5 are not separated and are electrophoresed as one band (the bands 6f in FIG. 4E, respectively). 6h and 6j). In addition, the band does not spread in the first and second migration directions more than expected by free diffusion.

On the other hand, the bands 6b and 6d composed of the candidate molecules 3b and 3d that bind to the target compound 5 are separated into a plurality of bands in the migration direction of the second electrophoresis. Alternatively, the band spreads in the second migration direction. That is, the band 6b is separated into a band 6 g composed of the candidate molecule 3b not bound to the target compound 5 and a band 6k composed of the candidate molecule 3b bound to the target compound 5 to form a complex. Alternatively, a wide band is formed between the bands 6g and the bands 6k, depending on the time that the candidate molecule 3 binds to the target compound 5. The band 6d of the candidate molecule 3d is similarly separated into a plurality of bands 6i and 6l, or a wide band is formed between the bands 6i and 6l.

Since the first and second electrophoresis are performed using the same electrophoresis method, the migration distances of the candidate molecules 3a, 3b, 3c, 3d and 3e in the first electrophoresis and the binding to the target compound 5 in the second electrophoresis The migration distances of the non-candidate molecules 3a, 3b, 3c, 3d and 3e are the same. Therefore, the bands 6f, 6g, 6h, 6i and 6j each consisting of the candidate molecules 3a, 3b, 3c, 3d and 3e that are not bound to the target compound 5 after the second electrophoresis are as shown in FIG. 4 (e). Line up in a straight line. Hereinafter, one straight line connecting such bands 6f, 6g, 6h, 6i and 6j will be referred to as a “straight line L”. If the migration direction of the first electrophoresis is the direction from the left side to the right side in FIG. 4 (e) and the migration direction of the second electrophoresis is the direction from the upper side to the lower side, the straight line L becomes a straight line with a downward slope to the right. ..

On the other hand, the bands 6k and 6l deviate from the straight line L because they are separated in the migration direction of the second electrophoresis. The migration distance of the band composed of the complex may be longer than the band 6g as in the band 6k or shorter than the band 6i as in the band 6l, depending on the target compound used and the type of electrophoresis method. ..

From the above, the candidate molecules 3b and 3d separated into a plurality of bands separated in the migration direction of the second migration on the second gel 8 after the second migration can be used as a molecular probe for capturing the target compound 5. Can be decided.

According to the method according to the second embodiment, since the two-dimensional electrophoresis is performed by the same electrophoresis method, it is possible to arrange the band composed of the candidate molecules not bound to the target compound 5 on the straight line L. .. Then, the band of the candidate molecule 3 bound to the target compound 5 is separated or spreads not in the longitudinal direction of the straight line L but in the migration direction of the second electrophoresis, so that it can be identified very easily. Therefore, even if the target compound is a low molecular weight compound, it can be easily identified that a plurality of bands are formed, and the molecular probe can be determined. In addition, such a method does not modify or fix the target compound, so that an appropriate molecular probe can be accurately determined. Therefore, even if the target compound is a low molecular weight compound, the molecular probe can be accurately determined.

The first electrophoresis and the second electrophoresis can be performed by any of the electrophoresis methods described in the first embodiment.

As the first gel 1, any gel described in the first embodiment selected according to the electrophoresis method used can be used. The second gel 8 is a gel having the same composition as the first gel 1. The first electrophoresis and the second electrophoresis may be continuously performed using one gel having a size capable of two-dimensional electrophoresis.

As the candidate molecule 3, any of the molecules described in the first embodiment can be used. Candidate molecules can be optically observably labeled as in the first embodiment. Alternatively, as in the first embodiment, the candidate molecule may be labeled after the electrophoresis.

One solution 4 can contain several to one million or more candidate molecules 3. For example, when the candidate molecule 3 is a nucleic acid aptamer, about 1 billion molecules are required for each type to be detected by fluorescent labeling, but one million types of nucleic acid sequences, that is, a total of 10 ^ 15 nucleic acids. If so, electrophoresis can be performed with one gel. Alternatively, since the straight line L can be detected even if the number of molecules per type is small, a maximum of 10 ^ 15 types of nucleic acid sequences can be included. This corresponds to the number of all combinations of 25 base long nucleic acid sequences. However, in this case, since the number of molecules in the band away from the straight line L is too small to be optically detected, the molecules are blindly recovered from the portion other than the straight line L. The recovered molecule is then amplified by a method such as PCR, and if necessary, electrophoresis is performed again. As the solvent of the solution 4, for example, the same solvent as in the first embodiment can be used. Solution 4 has a composition that does not contain substances that adversely affect each step of the method of the embodiment. Further, the solution 4 can have the same composition as the solvent when the candidate molecule 3 is used as a molecular probe.

As the reagent containing the target compound 5, the same reagent as in the first embodiment can be used. When the reagent is a liquid, the liquid reagent can be brought into contact with the region including the locations where the bands 6a, 6b, 6c, 6d and 6e are present on the first gel 1 after the first electrophoresis by dropping the liquid reagent. good. When the reagent is a gas, a container filled with the gas reagent containing a combination of the first gel 1 and the second gel 8 may be placed and the second electrophoresis may be performed. Alternatively, the second migration can be performed by putting the first gel 1 and the second gel 8 bound to each other in a container containing the target compound 5.

(Third Embodiment)
The method according to the third embodiment is a method for determining a molecular probe using a plurality of types of candidate molecules. In this example, the target compound is contacted with the candidate molecule prior to the first electrophoresis.

FIG. 5 is a diagram showing a schematic flow of a method for determining a molecular probe according to the third embodiment. The method includes the following steps. (S21) The target compound is brought into contact with a plurality of types of candidate molecules and electrophoresed on the gel (first electrophoresis), and (S22) the target compound is dissociated from the candidate molecules in the gel after the first electrophoresis. Electrophoresing a plurality of candidate molecules on the gel in a direction orthogonal to the migration direction of the first migration (second electrophoresis), and (S23) on the gel after the second electrophoresis, in the migration direction of the first electrophoresis. , The candidate molecule separated into a plurality of bands, or the candidate molecule whose band spreads in the migration direction of the first electrophoresis is determined as a molecular probe for capturing the target compound.

Hereinafter, the principle that a molecular probe that captures a target compound can be determined by performing each of the above steps will be described with reference to FIG. FIG. 6 is a schematic view showing a process of the method of the second embodiment.

First, a solution 4 containing a plurality of types of candidate molecules 3 is prepared. Solution 4 contains five candidate molecules 3a, 3b, 3c, 3d and 3e.

First, the solution 4 and the target compound 5 are added to a recess (not shown) formed at one end of the first gel 1 shown in FIG. 6 (a) (FIG. 6 (b)). Thereby, the candidate molecules 3a, 3b, 3c, 3d and 3e and the target compound 5 are brought into contact with each other. Part of the candidate molecules 3b and 3d that bind to the target compound 5 by contact binds to the target compound 5 to form complexes 7b and 7d, respectively. The other candidate molecules 3b and 3d remain unbound to target compound 5. Alternatively, a transition is made between 7b and 7d in the combined state and 3d and 3d in the unbonded state.

Then, the mixture added to the recess is electrophoresed (first electrophoresis) (FIG. 6 (c)). The migration direction of the first electrophoresis is the direction from the recess to which the solution 4 of the first gel 1 and the target compound are added toward the other end.

By the first electrophoresis, the candidate molecules 3a, 3b, 3c, 3d and 3e that do not bind to the target compound 5 are separated into bands 6a, 6b, 6c, 6d and 6e, respectively. Further, the complexes 7b and 7d are separated into bands 60b and 60d, respectively. Alternatively, the band spreads broadly between the bands 60b and 6b and between the bands 60d and 6d, depending on the time during which the complexes 7b, 7d are formed. The migration distance of the bands 60b and 60d may be longer than the band 6b as in the band 60b or shorter than the band 6d as in the band 60d, depending on the target compound used and the type of electrophoresis method.

Next, the target compound 5 is dissociated from the candidate molecules 3b and 3d in the first gel 1 ((d) in FIG. 6).

Next, as shown in FIG. 6 (e), the longitudinal side surface of the first gel 1 is bonded to the end of the second gel 8. Then, the electrophoresis process is performed in the direction orthogonal to the migration direction of the first electrophoresis (second electrophoresis). The second electrophoresis is performed using the same electrophoresis method as the first electrophoresis. The migration direction of the second electrophoresis is the direction from the end to which the first gel 1 is bound to the other end of the second gel 8.

By the second migration, the bands 6a, 6b, 6c, 6d and 6e, and the bands 60b and 60d move in the migration direction, respectively. Since the first and second electrophoresis are performed using the same electrophoresis method, the migration distances of the candidate molecules 3a, 3b, 3c, 3d and 3e that did not bind to the target compound 5 are the first electrophoresis and the first electrophoresis. It is the same as 2 electrophoresis. Therefore, the bands 6f, 6g, 6h, 6i and 6j after the second migration of the bands 6a, 6b, 6c, 6d and 6e, respectively, are aligned as shown in FIG. 4D.

On the other hand, the bands 60b and 60d were separated at different positions from the bands 6b and 6d in the first electrophoresis, but were composed of candidate molecules 3b and 3d that were not bound to the target compound 5 in the second electrophoresis. It is electrophoresed at the same distance as 6g and 6i. That is, it is electrophoresed to the positions of bands 6k and 6l in FIG. 6 (e).

Therefore, after the second migration, the candidate molecules 3b and 3d are separated into a plurality of bands in the first migration direction, respectively. Alternatively, if the bands 60b, 60d spread broadly between the bands 6b, 6d, the band after the second electrophoresis spreads broadly between 6g and 6k, and between 6l and 6i. Therefore, the candidate molecules 3b and 3d separated into a plurality of bands in the first migration direction or the bands spread in the first migration direction can be determined as the molecular probe for capturing the target compound 5.

According to the method according to the third embodiment, since the two-dimensional electrophoresis is performed by the same electrophoresis method, it is possible to arrange the band composed of the candidate molecules not bound to the target compound 5 in a straight line L shape. .. Then, since the band of the candidate molecule 3 bound to the target compound 5 is separated not in the longitudinal direction of the straight line L but in the migration direction of the first electrophoresis, it can be identified very easily. Therefore, it is possible to accurately determine the molecular probe of the desired target compound by a simple operation without modifying or immobilizing the target compound.

When the reagent containing the target compound 5 is a liquid, the reagent and the solution 4 may be mixed in advance and added to the first gel 1 before the step (S21). Alternatively, after adding the candidate molecule 3 to the first gel 1, a reagent may be further added thereto. When the reagent is a gas, the gas reagent may be blown into the solution 4 and added to the gel, or the first gel 1 may be placed in a container filled with the gas reagent to perform the first migration.

For the dissociation of the target compound 5 from the candidate molecule 3 in the step (S22), for example, after the first electrophoresis, the gel is left for a desired time or a reagent for dissociating the bond with the target compound is added to the first gel 1. It can be carried out by a method such as.

In a further embodiment, the method according to the second embodiment and the third embodiment is a step of identifying a candidate molecule determined to be a molecular probe after the step (S13) or (S23) of the method, respectively. (S14) or (S24) may be further included. The schematic flow of such a molecular probe determination method is shown in FIGS. 7 and 8.

For example, when a plurality of candidate molecules whose migration positions and arrangement orders are known are used, the migration distance of the band consisting of the molecular probe determined after the step (S13) or (S23) and the determined candidate molecules may be used. Candidate molecules may be identified from the order in which the bands are arranged. When identifying from the migration distance of the band, a standard reagent that forms a band that serves as an index of the migration distance may be used in the second migration. The standard reagents are electrophoresed together at positions on the second gel 8 that do not affect the band of candidate molecule 3.

Alternatively, when the position where the candidate molecules are migrated and the order in which the candidate molecules are arranged are unknown, the step (S14) or (S24) is performed as follows, for example. First, a gel piece containing a band consisting of a candidate molecule determined to be a molecular probe is cut out from the gel after the second electrophoresis. Next, a candidate molecule is extracted from the gel piece, and the candidate molecule is identified using any known analytical method selected according to the type of the candidate molecule.

For example, when the candidate molecule is a nucleic acid, the nucleic acid that is the candidate molecule is extracted from the gel piece, amplified, and sequenced. Alternatively, if it is a protein or peptide, a candidate molecule is extracted from the gel piece and analyzed by a known method for identifying the protein or peptide. When the candidate molecule is a protein or peptide, the identification step is more complicated than that of nucleic acid, so it is preferable to adopt the method of the first embodiment, which does not require identification of the candidate molecule.

When the candidate molecule 3 having a binding ability to the target compound 5 and the target compound 5 come into contact with each other, there is a candidate molecule 3 that does not bind to the target compound 5 with a very high probability as described above. However, it may be difficult to observe the band of the candidate molecule 3 in which most of the candidate molecules 3 are bound to the target compound 5 and are not bound to the target compound 5. A method for determining the molecular probe in such a case will be described with reference to FIG.

FIG. 9A shows the second gel 8 after the second electrophoresis of the step (S12) in the second embodiment. In this example, the case where most of the candidate molecules 3b bind to the target compound 5 and the band 6h composed of the candidate molecules 3b is difficult to observe is shown. In this case, the band 6k composed of the candidate molecule 3b bound to the target compound 5 exists at a position deviated from the straight line L connecting the unseparated bands 6f, 6h and 6j in the migration direction of the second electrophoresis. In this way, the candidate molecule 3 that produces a band deviating from the straight line L can be determined as the molecular probe.

FIG. 9B is a diagram showing a second gel 8 after the second electrophoresis of the step (S22) in the third embodiment. Also in this example, the candidate molecule 3 that produces a band deviating from the straight line L connecting the unseparated bands 6f, 6h, and 6j in the migration direction of the first electrophoresis can be determined as the molecular probe.

The straight line L can be determined as, for example, a straight line connecting two or more bands (for example, 6f, 6h or 6j) that are not separated. Alternatively, even if the candidate molecule binds to the target compound, a band may appear at a migration distance that does not bind to the target compound, and that band may be used to determine the straight line L.

Further, when a large number of types of candidate molecules are used in the methods of the second and third embodiments, the bands composed of the candidate molecules 3 that do not bind to the target compound 5, that is, are not separated, are lined up without gaps after the second electrophoresis. It may itself be observed as a straight line.

An example of such is shown in FIG. In that case, the candidate molecule 3b contained in the band 6k observed at a position deviating from the observed straight line 9 on the second gel 8 can be determined as a molecular probe for capturing the target compound. The band 6k deviates in the second migration direction in the case of the second embodiment, and deviates in the first migration direction in the case of the third embodiment. Alternatively, similarly to the above embodiment, the band 6k spreads as a straight line in the second migration direction in the case of the second embodiment according to the distribution of the binding time between the candidate molecule 3b and the target compound 5, and the third embodiment. In the case of, it spreads as a straight line in the first migration direction. Therefore, it is possible to identify the candidate molecule 3 bound to the target compound 5 even if many candidate molecules 3 are used at one time.

In the above embodiment, it has been explained that the molecular probe is determined, but it does not necessarily mean that the molecular probe is finally determined. For example, a plurality of candidate molecules may be extracted as promising candidates for a molecular probe. In that case, other analysis methods and evaluation methods may be carried out as necessary, and a candidate molecule that causes a larger change or a candidate molecule that causes a clearer change is selected again by the method of the above-mentioned example. It may be. When the candidate molecule is a nucleic acid, it may be amplified by a method such as PCR, and then the candidate of the molecular probe may be determined again by the method of the above-mentioned example. Alternatively, other types of candidate molecules may be narrowed down to a small number of candidate molecules in the second or third embodiment, and then finally determined in the first embodiment.

Further, when the candidate molecule is a single-stranded nucleic acid, the three-dimensional structure is unstable, and the band may be separated or spread even in the absence of the target compound. Such a phenomenon is not preferable because it may cause noise even when the candidate molecule is used as a molecular probe. Therefore, for example, conditions where separation or spread does not occur (for example, temperature, salt concentration, pH, etc.). Can be determined in advance in the absence of the target compound, and then the molecular probe can be determined by the method of the above embodiment. In addition, the conditions confirmed in advance here can also be used as conditions for detecting the target compound as a molecular probe. Alternatively, it is also possible to delete the candidate molecule whose three-dimensional structure is unstable in advance. For example, two-dimensional electrophoresis may be performed in the absence of a targetable substance in advance, and only the candidate molecules lined up on the straight line L may be recovered and the electrophoresis of the present embodiment may be performed.
The inventions described in the claims of the original application of the present application are described below.
[1]
A method of determining a molecular probe that captures a target compound.
(I) The target compound is brought into contact with one type of candidate molecule, and the obtained mixture is electrophoresed on a gel, and (ii) the candidate molecule has a plurality of bands on the gel after the electrophoresis. A method for determining a molecular probe, which comprises determining the candidate molecule as a molecular probe that captures the target compound when separated into.
[2]
A method of determining a molecular probe that captures a target compound.
(I) Electrophoresis treatment of a plurality of types of candidate molecules on a gel (first electrophoresis),
(Ii) The target compound is brought into contact with the plurality of types of candidate molecules on the gel after the first electrophoresis, and the obtained mixture on the gel is electrophoresed in a direction orthogonal to the electrophoresis direction of the first electrophoresis. Processing (second electrophoresis) and (iii) On the gel after the second electrophoresis, the candidate molecule separated into a plurality of bands in the migration direction of the second electrophoresis captures the target compound. Including determining with a molecular probe
The first electrophoresis and the second electrophoresis are methods for determining a molecular probe performed by the same electrophoresis method.
[3]
In the step (iii), the candidate molecule separated into the plurality of bands is the candidate molecule deviating from the straight line connecting the bands composed of the unseparated candidate molecules in the migration direction of the second electrophoresis [2]. ] The determination method described in.
[4]
A method of determining a molecular probe that captures a target compound.
(I) Contacting the target compound with a plurality of types of candidate molecules and electrophoresing the obtained mixture on a gel (first electrophoresis).
(Ii) Dissociating the target compound from the candidate molecule in the gel after the first electrophoresis, and electrophoresing the plurality of candidate molecules on the gel in a direction orthogonal to the migration direction of the first electrophoresis. (Second electrophoresis) and (iii) On the gel after the second electrophoresis, the candidate molecule separated into a plurality of bands in the migration direction of the first electrophoresis is a molecular probe that captures the target compound. Including deciding
The first electrophoresis and the second electrophoresis are methods for determining a molecular probe performed by the same electrophoresis method.
[5]
In the step (iii), the candidate molecule separated into the plurality of bands is the candidate molecule deviating from the straight line connecting the bands composed of the unseparated candidate molecules in the migration direction of the first electrophoresis [4]. ] The determination method described in.
[6]
The method according to any one of [2] to [5], further comprising identifying the candidate molecule determined to be the molecular probe.
[7]
The method according to any one of [1] to [6], wherein the target compound is a low molecular weight compound.
[8]
The method according to any one of [1] to [7], wherein the target compound is a gas.
[9]
The method according to any one of [1] to [8], wherein the candidate molecule is labeled with a fluorescent dye, a dye or a radioisotope.
[10]
The method according to any one of [1] to [9], wherein the electrophoresis is performed by isoelectric focusing or native electrophoresis.

1 ... 1st gel 3 ... Candidate molecule 4 ... Solution 5 ... Target compound 6a, 6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, 6j, 6k, 6l ... Band 8 ... Second gel 60b, 60d ... band

Claims (9)

A method of determining a molecular probe that captures a target compound.
(I) Electrophoresis treatment of a plurality of types of candidate molecules on a gel (first electrophoresis),
(Ii) The target compound is brought into contact with the plurality of types of candidate molecules on the gel after the first electrophoresis, and the obtained mixture on the gel is electrophoresed in a direction orthogonal to the migration direction of the first electrophoresis. Processing (second electrophoresis) and (iii) On the gel after the second electrophoresis, the candidate molecule separated into a plurality of bands in the migration direction of the second electrophoresis captures the target compound. Including determining with a molecular probe
The first electrophoresis and the second electrophoresis are methods for determining a molecular probe performed by the same electrophoresis method. Claim that the candidate molecule separated into the plurality of bands in the step (iii) is the candidate molecule deviating from the straight line connecting the bands composed of the unseparated candidate molecules in the migration direction of the second electrophoresis. The determination method according to 1. A method of determining a molecular probe that captures a target compound.
(I) Contacting the target compound with a plurality of types of candidate molecules and electrophoresing the obtained mixture on a gel (first electrophoresis).
(Ii) Dissociating the target compound from the candidate molecule in the gel after the first migration, and electrophoresing the plurality of candidate molecules on the gel in a direction orthogonal to the migration direction of the first migration. (Second electrophoresis) and (iii) On the gel after the second electrophoresis, the candidate molecule separated into a plurality of bands in the migration direction of the first electrophoresis is a molecular probe that captures the target compound. Including deciding
The first electrophoresis and the second electrophoresis are methods for determining a molecular probe performed by the same electrophoresis method. Claim that the candidate molecule separated into the plurality of bands in the step (iii) is the candidate molecule deviating from the straight line connecting the bands composed of the unseparated candidate molecules in the migration direction of the first electrophoresis. The determination method according to 3. The method according to any one of claims 1 to 4, further comprising identifying the candidate molecule determined to be the molecular probe. The method according to any one of claims 1 to 5, wherein the target compound is a low molecular weight compound. The method according to any one of claims 1 to 6, wherein the target compound is contained in a gas. The method according to any one of claims 1 to 7, wherein the candidate molecule is labeled with a fluorescent dye, dye or radioisotope. The method according to any one of claims 1 to 8, wherein the electrophoresis is performed by isoelectric focusing or native electrophoresis.

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